Use of GM-CSF as a vaccine adjuvant

ABSTRACT

The present invention is a method for enhancing the immune response of a mammal to a vaccine by administering to such a mammal an effective amount of GM-CSF in conjunction with a vaccine. The present invention further provides for pharmaceutical compositions containing an effective amount of GM-CSF and a vaccine. Kits containing GM-CSF and a vaccine are also disclosed.

This is a U.S. national application corresponding to InternationalApplication No. PCT/US93/06298, filed Jul. 7, 1993 designating the U.S.,which PCT application is a continuation-in-part of U.S. application Ser.No. 07/910,399, filed Jul. 8, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the use ofgranulocyte-macrophage-colony stimulating factor (GM-CSF), particularlyhuman GM-CSF, as a vaccine adjuvant.

BACKGROUND OF THE INVENTION

Active immunization is the administration of an antigen to an animal tobring about an immune response in the animal. A vaccine against amicroorganism is an antigenic preparation which when inoculated into anon-immune individual will confer active immunity to the microorganismbut will not cause disease. Specificity and memory, the two key elementsof the adaptive immune system, are exploited in vaccination, since theadaptive immune system mounts a much stronger response on secondencounter with an antigen. This secondary immune response is both fasterto appear and more effective than the primary response. The principle ofvaccine development is to alter a microorganism or its toxins (naturalantigens) in such a way that they become innocuous without losingantigenicity. Alternatively, antigenic polypeptides of the organism inquestion can be produced by recombinant methods or by syntheticchemistry to produce an effective vaccine.

One problem that frequently is encountered in the course of activeimmunization is that the antigens used in the vaccine are notsufficiently immunogenic to raise an antibody titer to sufficient levelsto provide protection against subsequent challenge, or to maintain thepotential for mounting these levels over extended time periods. Anotherproblem is that the vaccine may be deficient in inducing cell-mediatedimmunity which is a primary immune defense against bacterial and viralinfection. Still another problem is that an individual patient might beimmunosuppressed.

To obtain a stronger humoral and/or cellular response, it is common toadminister a vaccine in a formulation containing an adjuvant. Anadjuvant is a substance that enhances, nonspecifically, the immuneresponse to an antigen, or which causes an individual to respond to anantigen who would otherwise without the adjuvant not respond to theantigen. An adjuvant is usually administered with an antigen, but mayalso be given before or after antigen administration. Suitable adjuvantsfor the vaccination of mammals include but are not limited to Adjuvant65 (containing peanut oil, mannide monooleate and aluminummonostearate); Freund's complete or incomplete adjuvant; mineral gelssuch as aluminum hydroxide, aluminum phosphate and alum; surfactantssuch as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecyl-ammonium bromide,N,N-dioctadecyl-N',N'-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The antigens could also be administered following incorporation intoliposomes or other microcarriers.

SUMMARY OF THE INVENTION

It has been surprisingly discovered that granulocyte-macrophage-colonystimulating factor (GM-CSF) is an effective vaccine adjuvant.

Accordingly, the present invention provides a method for enhancing theimmune response of a mammal to a vaccine comprising administering to amammal in need of vaccination an effective amount of GM-CSF inconjunction with a vaccine.

Preferably, the mammals treated will be humans and the GM-CSF utilizedwill be one of the human allotypes. Preferably, the GM-CSF will beadministered from 1 to 14 days prior to or after the administration ofthe vaccine in an amount of about 0.1 to 100 micrograms (μg) perkilogram of body weight.

The present invention further provides for a pharmaceutical compositioncomprising an effective amount of GM-CSF, a natural, synthetic orrecombinant antigen, and a pharmaceutically acceptable carrier.

Also claimed is a kit for enhancing an immunogenic response of a mammalto antigens in a vaccine comprising a first container having apharmaceutical composition of GM-CSF contained therein; and a secondcontainer having a pharmaceutical composition of a vaccine containedtherein.

DETAILED DESCRIPTION OF THE INVENTION

The teachings of the references cited in the present application areincorporated herein in their entirety by reference.

According to the present invention, we have surprisingly found that theimmune response in a mammal, especially a human, to a vaccine can beeffectively enhanced by the administration of an effective amount ofGM-CSF in conjunction with the vaccine. The term "in conjunction with"as used herein refers to the administration of GM-CSF concurrently,before or following administration of a vaccine.

As used herein, "GM-CSF" means a protein which (a) has an amino acidsequence that is substantially identical to the sequence of mature(i.e., lacking a signal peptide) human GM-CSF described by Lee et al.Proc. Natl. Acad. Sci. U.S.A. 82: 4360 (1985) and (b) has biologicalactivity that is common to native GM-CSF.

Substantial identity of amino acid sequences means that the sequencesare identical or differ by one or more amino acid alterations(deletions, additions, substitutions) that do not substantially impairbiological activity. Among the human GM-CSFs, nucleotide sequence andamino add heterogeneity have been observed. For example, both threonineand isoleucine have been observed at position 100 of human GM-CSF withrespect to the N-terminal position of the amino acid sequence. Also,Schrimsher et al. Biochem. J 247:195 (1987)! have disclosed a humanGM-CSF variant in which the methionine residue at position 80 has beenreplaced by an isoleucine residue. GM-CSF of other species such as miceand gibbons (which contain only 3 methionines) and rats are alsocontemplated by this invention. Recombinant GM-CSFs produced inprokaryotic expression systems may also contain an additional N-terminalmethionine residue, as is well known in the art. Any GM-CSF meeting thesubstantial identity requirement is included, whether glycosylated(i.e., from natural sources or from a eukaryotic expression system) orunglycosylated (i.e., from a prokaryotic expression system or chemicalsynthesis).

GM-CSF for use in this invention can be obtained from natural sources(U.S. Pat. No. 4,438,032; Gasson et al., supra; Burgess et al., supra;Sparrow et al., Wu et al., supra). GM-CSF having substantially the sameamino acid sequence and the activity of naturally occurring GM-CSF maybe employed in the present invention. Complementary DNAs (cDNAs) forGM-CSF have been cloned and sequenced by a number of laboratories, e.g.Gough et al, Nature, 309:763 (1984) (mouse); Lee et al., Proc. Natl.Acad. Sci. USA, 82:4360 (1985) (human); Wong et al., Science, 228:810(1985) (human and gibbon); Cantrell et al., Proc. Natl. Acad. Sci. USA,82 :6250 (1985) (human), Gough et al., Nature, 309:763 (1984) (mouse);Wong et al., Science, 228:810 (1985) (human and gibbon); Cantrell etal., Proc. Natl. Acad. Sci. U.S.A., 82:6250 (1985) (human).

GM-CSF can also be obtained from Immunex, Inc. of Seattle, Wash. andSchering-Plough Corporation of Kenilworth, N.J. and from GenzymeCorporation of Boston, Mass.

Adjuvant activity is manifested by a significant increase inimmune-mediated protection by development of an immune response in anindividual who otherwise would not respond at all to a vaccine.Enhancement of humoral immunity is typically manifested by a significantincrease in the titer of antibody raised to the antigen.

The methods of the present invention to provide administration of GM-CSFin conjunction with a vaccine has the following advantages. The totalantigenic load of vaccine to be administered may be reduced since lessantigen in the presence of GM-CSF would elicit an immunologic responseat least equivalent to that achieved by the administration of the normalamount of the vaccine. Since less antigen would be required pervaccination by administering GM-CSF in accordance with the presentinvention, the probability of undesirable side-effects associated withsome vaccines currently in use would be reduced.

The immune response of certain types of individuals who respond poorlyto vaccination would be enhanced by administering GM-CSF in conjunctionwith a vaccine. Types of individual who should benefit from the methodsof the present invention include (1) those types having impaired immuneresponsiveness, (2) those individuals who appear normal but who arenevertheless nonresponsive to certain vaccines as well as (3)individuals undergoing immunosuppressive therapies such as radiation andchemotherapy.

Thus, we have discovered an effective method for (1) enhancing aneffective primary immune response in mammals to antigens present in avaccine, (2) enhancing an effective level of antibodies in mammalsexposed to antigen in vaccines, and (3) enhancing a primary immuneresponse in mammals to antigens present in a vaccine wherein the immuneresponse by the mammal without the administration GM-CSF would not bestrong enough or fast enough to prevent disease.

The vaccines contemplated for use in accordance with the presentinvention include but are not limited to bacterial vaccines, toxoidvaccines (inactivated toxins) and viral vaccines or mixtures thereofused for active immunization. See for example chapter 75 entitled"Immunizing Agents" in Remington's Pharmaceutical Sciences 14th Edition1990 Mack Publishing Co. p 1426-1441 and the antitoxins, toxoids,vaccines and live vaccines approved by the U.S. Food and DrugAdministration and listed on page 208-209 (Product Category Index) ofthe Physician's Desk Reference, 46th Ed. 1992. Suitable bacterialvaccines include bacterial vaccines against the following diseaseentities or states: cholera, pertussis, plague, typhoid fever,meningitis, pneumococcal pneumonia, H. influenzae type B, leprosy,gonorrhea, Group B meningococcus, and Group B streptococcus,Gram-negative sepsis, E. coli sepsis, and Pseudomonas aeruginosa.Suitable toxoids include diphtheria toxoid, botulism toxid, and tetanustoxoid. Suitable vital vaccines include live and inactivated viralvaccines against the following disease entities or states:poliomyelitis, measles rubella, yellow fever, mumps, hepatitis B,hepatitis C and viral influenza.

The suitable "multiple antigens" include diphtheria and tetanus toxoids,the triple antigen-diphtheria, pertussis and tetanus toxoids such as areavailable from Connaught Laboratories, Inc. Swiftevater, Pa. 18370. Thewide variety of viral strains and cell substrates and the the variedimmunization schedules used in different countries are disclosed inWhite, D.O., and Fenner F., Medical Virology. 3rd Edition (AcademicPress 1986).

In addition, the GM-CSF will typically be used to enhance the protectionafforded by animal or human vaccines that are considered "weak" (i.e.,provide diminished protection in terms of level, extent, and/orduration). Examples of such vaccines are bacterins such as Bordetellabacterin, Escherichia coli bacterins, Haemophilus bacterins,Leptospirosis vaccines, Moraxella bovis bacterin, Pasteurella bacterinand Vibrio fetus bacterin, pneumococcal vaccines and attenuated live orkilled virus products or recombinant antigenic viral products such ashepatitis B, influenza A & B, bovine respiratory disease vaccine,infectious bovine rhinotracheitis, parainfluenza-3, respiratorysyncytial virus, bovine virus diarrhea vaccine, equine influenzavaccine, feline leukemia vaccine, feline respiratory disease vaccinerhinotracheitiscalicipneumonitis viruses, canine parovovirus vaccine,transmissible gastroenteritis vaccine, pseudorabies vaccine, and rabiesvaccine.

The term "effective amount" as used herein regarding the effectiveamount of GM-CSF administered in accordance with the present inventionmeans an amount of GM-CSF which produces an increase in antibody levelsufficient to provide increased protection from an infectious agent thanif a vaccine had been administered without GM-CSF. However, it should benoted a significant increase in antibody level may be relatively small.The effective amount of GM-CSF administered is from 0.1 to 500 μg ofGM-CSF per kilogram of body weight. More preferably, the effectiveamount administered is from 1 μg to 100 μg and most preferably from 5 to50 μg of GM-CSF per kilogram of body weight.

The amount, frequency and period of administration will vary dependingupon factors such as the level of the specific antibody titers, theclass of antibody to be induced, the vaccine type as well as the age ofthe patient and general physical condition. The GM-CSF can be 5administered before, concurrently with or after the vaccine isadministered. Preferably, one dose of GM-CSF is given to the patientfrom 1 to 14 days prior to the administration of the vaccine. Mostpreferably the GM-CSF is administered about 24 hours prior to or afterthe administration of the vaccine.

The GM-CSF will normally be administered separately from the vaccine,although it may be administered in combination with the vaccine. WhenGM-CSF is combined with the vaccine, the composition administeredcontains an immunogen that is effective in eliciting a specific responseto a given pathogen or antigen, a pharmaceutically acceptable vaccinecarrier and an immunopotentiating amount of GM-CSF. Administration ofGM-CSF can be subcutaneous, intravenous, parenteral, intramuscular, orany other acceptable method. Preferably, GM-CSF is administered prior tothe administration of the vaccine and at the same site where the vaccineis to be administered. The formulations and pharmaceutical compositionscontemplated by the above dosage forms can be prepared with conventionalpharmaceutically acceptable excipients and additives, using conventionaltechniques. Other adjuvants may be administered either with the vaccineor together with the GM-CSF.

If multiple doses of the vaccine are to be administered over a period oftime, additional GM-CSF may be administered in conjunction with eachsubsequent dose of the vaccine. The amount of GM-CSF which isadministered with each subsequent dose of the vaccine may be more, thesame or less than the amount of GM-CSF administered in conjunction withthe initial dose of the vaccine. The amount of GM-CSF administered witheach subsequent dose of the vaccine will depend upon the antibodyresponse of the patient after the first dose of the vaccine.

Solutions of GM-CSF to be administered may be reconstituted fromlyophilized powders and they may additionally contain preservativesbuffers, dispersants, etc. Preferably, GM-CSF is reconstituted with anyisotonic medium normally utilized for subcutaneous injection, e.g.,preservative-free sterile water.

A sustained release formulation of GM-CSF can be administered which willresult in a longer serum half-life of the drug. Examples of suchformulations are the following:

    ______________________________________                                        Formulation 1                                                                 INGREDIENTS                                                                   ______________________________________                                        Lyopilized GM-CSF    10-1000  mcg                                             Zinc Acetate         4.0      mg                                              Protamine Sulfate    2.5      mg                                              Sodium Hydroxide     0.6      mg                                              Water for Injection q.s.                                                                           1        ml                                              ______________________________________                                    

To prepare the sustained release preparation of GM-CSF according toFormulation 1, the lyophilized GM-CSF is dissolved in a portion of theWater for Injection and the pH of the solution is adjusted to 8.2 usingsodium hydroxide. The protamine sulfate is added and the mixture isagitated, after which the zinc acetate is added and the mixture is againagitated. The total solution is brought to the final volume with theremaining Water for Injection. Preferably, the sodium hydroxide,protamine sulfate and zinc acetate are added as concentrated aqueoussolutions (e.g. for protamine, 100 microliters of a 25 mg/ml aqueoussolution).

    ______________________________________                                        Formulation 2                                                                 INGREDIENTS                                                                   ______________________________________                                        Lyopilized GM-CSF      10-1000  mcg                                           Water-for-injection for reconstitution                                                               0.2      ml                                            Dioctyl Sodium Sulfosuccinate                                                                        1        mg                                            Peanut oil for emulsion                                                                              2        ml                                            Peanut oil for gel     2        ml                                            Aluminum monostearate  50       mg                                            ______________________________________                                    

To prepare the sustained release preparation of GM-CSF according toFormulation 2, the aluminum monostearate is mixed into the peanut oilfor the gel and heat elevated to form the gel according to knownmethods.

The dioctyl sodium sulfosuccinate is dissolved into the Water forInjection. The lyophilized GM-CSF is reconstituted with the dioctylsodium sulfosuccinate solution, the resultant solution is transferedinto the peanut oil for emulsion and mixed by vortexing. The resultantemulsion is then mixed into the previously prepared gelled peanut oiland mixed by vortexing.

    ______________________________________                                        Formulation 3                                                                 INGREDIENTS                                                                   ______________________________________                                        Lyopilized GM-CSF     10-1000  mcg                                            Copper Acetate        0.2      mg                                             Sodium Phosphate, Dibasic                                                                           2.27     mg                                             Sodium Phosphate, Monobasic                                                                         0.55     mg                                             Sodium Hydroxide      0.6      mg                                             Water for Injection q.s.                                                                            1        ml                                             ______________________________________                                    

To prepare the sustained release preparation of GM-CSF according toFormulation 3, the monobasic and dibasic sodium phosphates are dissolvedin a portion of the Water for Injection. The lyophilized GM-CSF is thendissolved in this solution and the pH is adjusted to 7.8 with the sodiumhydroxide. The copper acetate is then added and the solution isagitated. The solution is brought to final volume using the remainingWater for Injection. Preferably, the sodium hydroxide and copper acetateare added as concentrated aqueous solutions (e.g., for copper acetate,100 microliters of a 2 mg/ml aqueous solution).

Additional Sustained Release Formulations

Additional sustained release formulations of GM-CSF can be preparedusing micoencapsulated or microspheres of GM-CSF prepared using polymerssuch as polyanhydrides, polyphosphazenes, collagen, alginates,poly(methacrylates), gelatin, poly(hydroxybutyrate), poly(caprolactone),ethylene vinyl acetate or polylactide glycolide.

Sustained release GM-CSF can also be prepared as chemical conjugates ofGM-CSF using polyethylene glycol, dextran poly(aminoacids) and othersimilar polymers.

The effect of GM-CSF on enhancing the immune response of a vaccine isillustrated by the following non-limiting human clinical data whichshould not be construed to limit the scope of the disclosure.

EXAMPLE 1

Recombinant human GM-CSF was shown to enhance the efficacy ofrecombinant hepatitis B vaccine on dialysis patients who had notresponded to the hepatitis B vaccine.

The objective of the present experiment was to determine whether theco-administration of GM-CSF and hepatitis vaccine would be capable ofrestoring immunologic responsiveness to patients with renal failure whohad been previously unresponsive to hepatitis vaccination.

Fifteen dialysis patients who had not responded to at least 3 attemptsat vaccination with hepatitis B vaccine as determined by their antibodytiters against the hepatitis B surface antigen (HBsAg) were treated withGM-CSF. Six patients were injected subcutaneously with 0.5 micrograms(μg) of GM-CSF per kilogram (kg) weight of the patient, (produced in anE. coli. expression system by Scher/ng-Plough, Kenilworth, N.J., USA),five patients with 5 μg of GM-CSF per kg weight of the patient and fourpatients with 10 μg of GM-CSF per kg weight of the patient, and the siteof injection was marked. Twenty-four hours after administration of theGM-CSF, the patients were each administered 40 μg of the hepatitis Bvaccine, HBVax® (Merck, Sharpe and Dohme, Gmbh, Darmstadt, FederalRepublic of Germany) at the same site as the GM-CSF was injected. Fourweeks after administration of the vaccine, blood samples were drawn fromthe patients and the samples were tested for the presence ofanti-hepatitis B antibody. The results are shown in the table below.

                  TABLE 1                                                         ______________________________________                                        Dosage of GM-CSF                                                                            Antibody titer against HBsAg                                    (μg/kg wt.)                                                                              Units*/liter                                                    ______________________________________                                        0.5           0                                                               0.5           0                                                               0.5           0                                                               0.5           0                                                               0.5           0                                                               0.5           710                                                             5.0           0                                                               5.0           35                                                              5.0           125                                                             5.0           920                                                             5.0           2600                                                            10.0          0                                                               10.0          0                                                               10.0          440                                                             10.0          7240                                                            ______________________________________                                         *A unit is defined as the reciprocal of the serum dilution which produced     a half maximal response in a standard ELISA.                             

As can be seen the data presented above, GM-CSF was an effectiveadjuvant used in conjunction with the hepatitis B vaccine.

EXAMPLE 2

Recombinant human GM-CSF was shown to enhance the efficacy of viralinfluenza vaccine in elderly patients.

Human influenza viruses, occurring during pandemics (Influenza A) andepidemics (Influenza A and B), cause significant excess morbidity andmorality in the elderly, not only in those with underlying chronicdiseases, but also in apparently healthy subjects. Since influenzavaccine has been shown to provide benefits in reducing both morbidityand mortality, flu vaccine is strongly recommended in subjects at highrisk to develop flu related complications, and substantial resources areexpended annually in an effort to vaccinate high-risk subjects.

However, despite large immunization programs, influenza remains asignificant cause of illness and death in the elderly. Several methods,such as administering two to three times the standard vaccine dose orgiving a booster dose one month after a first standard dose have notbeen shown to improve immunoresponse to flu vaccine in the elderly.

Accordingly, a double-blind, placebo-controlled, dose escalation studywas carried out to determine whether the immunoresponse to flu vaccineis enhanced by the administration of recombinant GM-CSF. Five differentdosages of recombinant GM-CSF (produced in an E. coli expression systemby Schering-Plough, Kenilworth, N.J., USA) were tested, namely, 0.25,0.5, 1, 2.5 and 5 μg/kg in comparison to placebo. Sixty elderly healthysubjects were enrolled. They received recombinant GM-CSF or a placebosubcutaneously in one arm just before the intramuscular administrationof the French 1992-1993 trivalent flu vaccine (A/Singapore/6/86 H1N1!,A/Beijing/353/89 H3N2! and B/Yamagata/16/88) in the other arm (PasteurVaccins, Marnes-la-Coquette, France). Specific hemoagglutinin-inhibiting(HAI) antibody titers against the three flu virus strains weredetermined at baseline and 1, 3 and 6 weeks after vaccination. None ofthe 15 patients who received placebo with the flu vaccine showedsimultaneous seroconversion to all the three strains of the flu vaccine,whereas 5 (56%) and 3(33%) of the 9 patients receiving 2.5 and 5 μg/kgof recombinant GM-CSF seroconverted to all three strains.

The protocol which was used is described in more detail below.

Materials and Methods

Subject Selection. Subjects were healthy elderly people of both sexesand of at least 65 years of age. Volunteers were screened by medicalhistory, and laboratory tests that included a complete blood cell count,biochemistry, urinalysis and serology. Subjects had to have a baselinehemoagglutinin-inhibiting (HAI) antibody titers ≦1:40 for the A-H1N1 andB influenza strains and ≦1:80 for the A-H3N2 influenza strain containedin the 1992-1993 French flu vaccine. Subjects with history of severe orunstable chronic illness or malignancy, taking antineoplastic orimmunosuppressive drugs, with significantly abnormal results of thescreening laboratory tests, allergic to eggs, with history of aninfluenza like illness in the past six months, or acutely ill at thetime of specimen collection were excluded from the study.

Study Design. The study was designed as a double-blind,placebo-controlled, dose finding study.

Drug administration and vaccination. The 60 subjects enrolled in thestudy were divided into five dose groups of 12 subjects each. Each groupreceived a single dose of 0.25, 0.5, 1, 2.5 or 5 μg/kg of r GM-CSF orplacebo. Recombinant GM-CSF or placebo were administered subcutaneouslyin the deltoid area of the right arm, immediately after which all thesubjects received 0.5 ml of a licensed 1992-1993 trivalent subvirionvaccine that contained 15 μg each of HAs from A/Singapore/6/86 (H1N1),A/Beijing/353/89 (H3N2) and B/Yamagata/16/88 viruses (Pasteur Vaccins,Marnes-la-Coquette, France) intramuscularly in the deltoid of the leftarm.

Specimen collection. Blood serum and throat swabs for the virologicalcultures were obtained from each subject before the start of the studyand 1,3 and 6 weeks after vaccination.

Serum antibodies. HAI antibodies to influenza A/Singapore (NH1N1),A/Beijing (H3N2) and B/Yamagata virus antigens were measured in serumspecimens by a standard hemo-agglutination inhibition assay. Results forthe preliminary analysis of the sera collected until Week 6 wereobtained by testing all specimens on the same day using identicalreagents. The initial starting dilution was 1:20. HAI antibody titersless than 1:20 were defined as "Not detectable" titers.

StatisticaI analysis. Success according to the antibody titers weredefined in two different ways:

Seroconversion: a four-fold increase of the HAI antibody titers overbaseline at Week 6.

Seroprotection: HAI antibody titers greater than baseline and at leastequal to 1:40 at Week 6.

The number of successes based on these definitions were analyzed usingthe Fisher's exact tests. The placebo groups of the different dosegroups were pooled together.

Results

Subject characteristics. All the 60 healthy elderly volunteers enrolledin the study completed the assessment at the 6th week. Table 3 reportsHAI antibody titers at baseline. Most of the subjects had baseline HAIantibody titers below protective levels, that is below 1:40.Distribution of the baseline titers was similar among all treatmentgroups, with the exception of the group treated with 0.5 μg/kg in whichmost of the patients had no detectable antibody titers.

Immunoresponse. Table 4 shows the number of patients who seroconvertedto all three strains 6 weeks after the administration of GM-CSF/placeboand flu vaccine. Results are also reported separately for each strain.

None of the 15 subjects who received placebo with the flu vaccine showedsimultaneous seroconversion to all three flu virus strains, whereas 5 of9 (56%) and 3 of 9 (33%) of the subjects treated with 2.5 and 5 μg/kg ofthe rGM-CSF were seroconverted to all three strains. In addition, whenthe results are examined separately for each flu strain, seroconversionrates for 2.5 and 5 μg/kg of rGM-CSF are consistently higher (rangingfrom 44% to 67%) than those observed with placebo (ranging from 13% to20%). Interestingly, seroconversion rates observed with the lowest dose(0.25 μg/kg) have been higher than those observed with 0.5 and 1 μg/kg.

As shown in Table 5, the conclusion of the results do not change alsousing "seroprotection" as definition of success. As expected,seroprotection rates are higher than those of seroconversion in alltreatment groups, because seroprotection is easier to accomplish thanseroconversion. Again, patients treated with rGM-CSF showed a higherimmunoresponse rate to the flu vaccine than those treated with placebo.In fact, 5 of 9 (56%) and 3 of 9 (33%) patients treated with 2.5 and 5μg/kg were protected against all three strains, whereas only 1 of the 15subjects (7%) treated with placebo was seroprotected against all threestrains.

                  TABLE 2                                                         ______________________________________                                        Study design.                                                                                Number of subjects                                                            CSF 39300                                                                             Placebo                                                ______________________________________                                        Group I: 0.25 μg/kg                                                                         9         3                                                  Group II: 0.5 μg/kg                                                                         9         3                                                  Group III: 1. μg/kg                                                                         9         3                                                  Group IV: 2.5 μg/kg                                                                         9         3                                                  Group V: 5. μg/kg                                                                           9         3                                                  ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    HAI antibody titers at baseline. The table reports the number of subjects     in each treatment                                                             group according to baseline HAI antibody titers for each of the three flu     strains.                                                                                   Number of subjects with specific HAI antibody titers at                       baseline                                                                      A/Beijing   A/Singapore                                                        H3N2!       H1N1!   B/Yamagata                                  Baseline HAI antibody titers:                                                              <1:20                                                                            1:20                                                                             1:40                                                                             1:80                                                                             <1:20                                                                            1:20                                                                             1:40                                                                             <1:20                                                                            1:20                                                                             1:40                                  __________________________________________________________________________    Placebo + flu vaccine                                                                      5  6  4  -- 10 4  1  10 2  3                                     CSF 39300 + flu vaccine                                                       0.25 μg/kg                                                                              4  3  2  -- 6  1  2  5  4  --                                     0.5 μg/kg                                                                              1  5  3  -- 7  1  1  4  1  4                                       1 μg/kg 2  4  3  -- 4  3  2  4  2  3                                      2.5 μg/kg                                                                              2  4  1  2  6  2  1  4  5  --                                      5 μg/kg 9  -- -- -- 8  1  -- 8  1  --                                    __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Results of seroconverstion.                                                   Seroconversion is defined as a four-fold increase of the HAI antibody         titers over baseline. The second column of the table reports the number       of subjects who                                                               seroconverted to all three strains contained in the flu vaccine. The last     three columns show the                                                        number of seroconversion observed for each flu strain.                                                Number of seroconversion observed                                  Patients simultaneously                                                                  for the diferent flu strains                                       seroconverted to all                                                                     A/Beijing                                                                           A/Singapore                                                  three strains                                                                             H3N2!                                                                               H1N1!                                                                              B/Yamagata                                __________________________________________________________________________    Placebo + flu vaccine                                                                      0/15 (0%)  3/15 (20%)                                                                          3/15 (20%)                                                                          2/15 (13%)                                CSF 39300 + flu vaccine                                                       0.25 μg/kg                                                                              2/9 (22%)  4/9 (44%)                                                                           4/9 (44%)                                                                           3/9 (33%)                                  0.5 μg/kg                                                                              0/9 (0%)   2/9 (22%)                                                                           3/9 (33%)                                                                           1/9 (11%)                                   1 μg/kg 1/9 (11%)  2/9 (22%)                                                                           1/9 (11%)                                                                           4/9 (44%)                                  2.5 μg/kg                                                                              5/9 (56%)  6/9 (67%)                                                                           5/9 (56%)                                                                           5/9 (56%)                                   5 μg/kg 3/9 (33%)  4/9 (44%)                                                                           4/9 (44%)                                                                           5/9 (56%)                                 __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Results of seroprotection. Seroprotection is defined as an increase of        the HAI antibody titers over                                                  baseline and at least 1:40. The second column of the table reports the        number of subjects who develop                                                seroprotective HAI antibody titers against all three strains contained in     the flu vaccine.                                                              The last three columns show the number of seroprotection observed for         each flu strain.                                                                                      Number of seroprotection observed                                  Patients simultaneously                                                                  for the diferent flu strains                                       seroprotected to all                                                                     A/Beijing                                                                           A/Singapore                                                  three strains                                                                             H3N2!                                                                               H1N1!                                                                              B/Yamagata                                __________________________________________________________________________    Placebo + flu vaccine                                                                      1/15 (7%)  8/15 (53%)                                                                          5/15 (33%)                                                                          3/15 (20%)                                CSF 39300 + flu vaccine                                                       0.25 μg/kg                                                                              4/9 (44%)  6/9 (67%)                                                                           5/9 (56%)                                                                           6/9 (67%)                                  0.5 μg/kg                                                                              1/9 (11%)  5/9 (56%)                                                                           3/9 (33%)                                                                           3/9 (33%)                                   1 μg/kg 2/9 (22%)  4/9 (44%)                                                                           3/9 (33%)                                                                           4/9 (44%)                                  2.5 μg/kg                                                                              5/9 (56%)  8/9 (89%)                                                                           5/9 (56%)                                                                           6/9 (67%)                                   5 μg/kg 3/9 (33%)  4/9 (44%)                                                                           4/9 (44%)                                                                           5/9 (56%)                                 __________________________________________________________________________

What is claimed is:
 1. A method for enhancing an immune response of amammal to a vaccine comprising administering to a mammal in need ofvaccination an effective amount of granulocyte-macrophage-colonystimulating factor (GM-CSF) in conjunction with a vaccine, saideffective amount of GM-CSF being sufficient to enhance an immuneresponse to said vaccine.
 2. The method of claim 1 wherein the vaccineis selected from a group consisting of hepatitis B vaccine and influenzavaccine.
 3. The method of claim 1 wherein the GM-CSF which isadministered is contained within a sustained release formulation.
 4. Themethod of claim 1 wherein the immune response is an antibody-mediatedimmune response.
 5. The method of claim 1 wherein said mammal is ahuman.
 6. The method of claim 4 wherein said mammal is a human.
 7. Apharmaceutical composition comprising an effective amount of GM-CSF anda vaccine, said effective amount of GM-CSF being sufficient to enhancean immune response to said vaccine.
 8. The pharmaceutical composition ofclaim 7 wherein the GM-CSF is contained within a sustained releaseformulation.
 9. A kit for enhancing an immunogenic response of a mammalto antigens in a vaccine comprising a container of a pharmaceuticalcomposition of GM-CSF and a pharmaceutically acceptable carriertherefor; and a container of a pharmaceutical composition of a vaccineand a pharmaceutically acceptable carrier therefor.
 10. The kit of claim9 wherein the GM-CSF is contained within a sustained releaseformulation.